KR20140052941A - Method for detectinon of intestinal, and blood-brain barrier permeability and testing materials thereto - Google Patents

Abstract

The present invention relates to a method, an assay method and an apparatus for testing for intestinal and / or blood-brain barrier permeability related antigen. For example, blood, saliva, or other bodily fluids may contain at least one of (1) a binding to a bacterial toxin (preferably a lipopolysaccharide) and (2) ≪ / RTI > is tested for binding to selected tissue antigens. The test results are used to support the detection and diagnosis of diseases associated with intestinal leak syndrome (whether due to intracellular or intracellular pathways, bacterial toxins or other causes) and / or excessive blood brain barrier permeability related diseases, Is used for the diagnosis of neuroinflammation and / or neuronal autoimmune diseases, in particular, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, Alzheimer's disease, or peripheral neuropathy, major depression.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for detecting blood-brain barrier permeability,

This application claims the benefit of U.S. Provisional Application No. 61 / 437,244 filed on January 28, 2011, which is incorporated by reference in its entirety. The present invention relates to a method, an analysis apparatus and a kit for detecting and diagnosing bowel and blood-brain barrier permeability.

Compared to other human organelles, epithelial cells are exposed to a large number of antigens from food, yeast, bacteria and viruses. Some bacterial antigens are not a threat to the mucosal immune system, but others can be harmful to the host. The intestinal immune system monitors these bacterial antigens in the intestinal lumen by allowing small amounts of molecules to penetrate epithelial cells that interact with mucosal and systemic immune systems to trigger regulatory T cell function or resistance to these antigens . However, inadequate or overexpression of the intestinal immune system in these bacterial antigens can lead to the disruption of the regulatory mechanism and can lead to gastrointestinal disease (1). Thus, the physiological understanding of antigen uptake is crucial to understanding disease pathologies, including inflammation and autoimmune reactions (2).

These and all other extraneous materials discussed herein are incorporated herein by reference in their entirety. Where the definition or use of a term in an incorporated reference is inconsistent or is in conflict with a definition provided herein, the definitions provided herein apply and the definitions of the terms in the reference do not apply.

Increased intestinal permeability is understood to be an early step leading to the onset of multiple autoimmune diseases (3-6). For these reasons, recent interest in the role of the intestinal barrier function has been increasing in various pathological conditions of the extra-organ such as the nervous system as well as the gastrointestinal tract (7). Control abnormalities of intestinal barrier function as a biological entry into inflammation, autoimmunity, and cancer have been discussed in the Fasano document (7). In this document, as well as the previous Fasano document (4), Fasano emphasized that the main function of the gastrointestinal tract has traditionally been limited to digestion and nutrient uptake and electrolytes and water homeostasis. A more detailed analysis of the gastrointestinal anatomical and functional configuration suggests that another very important function of this institution is to control the transport of macromolecules between the environment and the host via a barrier mechanism. With intestinal-associated lymphoid tissue and neuroendocrine networks, intestinal epithelial barrier regulates the balance between immune tolerance and immune responses to intercellular fusion membranes and self-antigens.

Crude / aocludin is a physiological modulator of the tight junctions involved in macromolecular transport, thus establishing a balance between immune response and tolerance (7). If the intestinal barrier protein that is finely regulated in a genetically vulnerable individual in an environmental factor is regulated, then an intestinal disorder such as chronic digestive dysfunction, Crohn's disease and ulcerative colitis, and an extrinsic disease such as an autoimmune disease arthritis, lupus, thyroid , diabetes. And even autoimmune diseases such as multiple sclerosis (MS), malignant tumors and major depression (8-14). One of the important environmental factors involved in the pathophysiology of intestinal and brain barrier dysfunction, and therefore in intestinal and extra-amniocentesis, is bacterial lipopolysaccharide (LPS). Due to intestinal bacterial imbalance and bacterial migration of intestinal microflora, LPS contributes to the activation of the toll-like receptors and inflammatory responses of epithelial cells, which in turn lead to blood-brain barrier function abnormalities after the intestinal barrier (14). The role of LPS in inducing "bowel leak" and "brain leak" syndrome is shown in FIG.

Figure 1 highlights that the GI tract abnormality is a threat to the immune system by compromising intestinal barrier integrity and thus increasing the entry of non-digested antigens into the blood circulation. The response to these antigens is to activate the immune and inflammatory responses leading to inflammatory cytokines, antibody array production and increased intestinal barrier permeability (or "intestinal leak" syndrome). If intestinal wall control abnormalities remain untreated, the results may lead to nerve inflammation, nerve infiltration, and neural degeneration.

There is therefore a need for non-invasive methods, devices, and analyzers for the determination of intestinal permeability of large antigen molecules that are open to the immune system, including the inflammation that leads to blood-brain barrier opening and subsequent inflammation and neural degeneration . The present invention is directed to these and other needs.

The present invention provides devices, systems, analyzers and methods that can be tested to assist in the detection and diagnosis of intestinal and / or blood-brain barrier permeability.

In certain aspects of the invention, one or more portions of the sample comprise at least one of the following: (1) a binding to a bacterial toxin; and (2) a natural antigen selected from at least one of (a) Lt; / RTI > is tested. In some aspects, the bacterial toxin advantageously comprises a lipid polysaccharide.

When testing intestinal permeability, the natural intestinal-associated antigen is preferably selected from (1) intestinal structural proteins; (2) fusion membrane proteins; (3) a binding receptor for a fusion membrane protein; And (4) cell boundary proteins. In an embodiment of certain aspects of the present invention there is provided a method of inhibiting proliferation of at least one of actin / actomyosin, acrolein and / or choline, ZOT receptors and matrix metalloproteinase-3 (MMP-3) Antibody testing is in progress.

When testing the permeability of the blood brain barrier, the blood brain barrier-associated antigen is preferably (1) a blood brain barrier protein; (2) gliophage fibrous acid protein (GFAP); (3) matrix metalloproteinase (MMP); (4) brain ZOT binding protein; (5) brain ZOT receptor; (6) Calprotectin; And (7) a herbal basic protein. In an embodiment of certain aspects of the present invention there is provided a composition comprising (1) a blood brain barrier protein; (2) gliophage fibrous acid protein (GFAP); (3) an antibody test for at least one of the substrate metalloproteinases (MMPs).

From a diagnostic point of view, analysis of one or more of the above described methods can be applied to disease detection and / or diagnostic assays related to intestinal leak syndrome and / or permeability of excessive blood brain barrier.

In some aspects of the invention, binding sample detection for each component is performed by immunoassay and includes, but is not limited to, ELISA analysis, RIA analysis, latex aggregation, bead analysis, protein analysis and other immunological assays known to those skilled in the art do.

In a particular aspect of the invention, a test plate and kit for immunoassay is provided wherein the improved test plate comprises, for example, (1) a bacterial toxin as a binding peptide, and (2) ) Blood natural brain barrier-associated antigen.

In particularly preferred test plates used to detect and diagnose diseases associated with intestinal leak syndrome or to assist in identification, enteric-associated antigens are preferably (1) intestinal structural proteins; (2) fusion membrane proteins; (3) a binding receptor for a fusion membrane protein; And (4) cell boundary proteins.

In a test plate that is particularly suitable for diagnosing or identifying diseases associated with excessive blood brain barrier permeability, the blood brain barrier-associated antigen preferably comprises (1) a blood brain barrier protein; (2) gliophage fibrous acid protein (GFAP); (3) matrix metalloproteinase (MMP); (4) brain ZOT binding protein; (5) brain ZOT receptor; (6) Calprotectin; And (7) a herbal basic protein.

Test kits are contemplated that include one or more plates that can be collectively tested for both the antigen of the first set associated with the intestinal leak syndrome and the antigen of the second set associated with excessive blood brain barrier permeability.

In a more general aspect, a method and apparatus for supporting the detection and diagnosis of diseases associated with excessive permeability of anatomical barriers is contemplated herein, wherein the patient sample comprises a bacterial toxin, and (a) a enteric- And obtaining and analyzing test results from an antibody test panel that generates a signal due to binding with natural antigens selected from at least one of the blood natural brain barrier-associated antigens.

In all of these methods and apparatus contemplated herein, the sample includes any suitable body sample, including, for example, a whole blood sample, a serum sample, a saliva sample, or other body fluid sample.

The methods and apparatus contemplated herein may be applied to differential diagnosis of diseases related to (1) intestinal bacterial imbalance of intestinal microbial guns, and (2) intestinal wall collapse. For example, the diagnosis of intestinal bacterial imbalance in intestinal microbial counts is based on the test results that any IgA, IgM, and IgG results for bacterial toxins of lipid polysaccharides are all IgA, IgM, and IgM for positive, IgG results are negative, and IgG results for actomyosin are negative.

It can also assist in the diagnosis of intestinal wall decay due to the peri-cellular and trans-cellular pathways.

In connection with the pericellular pathway disruption, diagnosis of intestinal wall decay by bacterial antigens is based on the test results that any IgA, IgM, and IgG results for bacterial toxins of lipopolysaccharides are positive for any positive, IgA, IgM, and IgG results are positive, and IgG results for actomyosin are negative. Conversely, intestinal barrier degradation-related diagnoses other than bacterial antigens were tested, as a result of testing, that all IgA, IgM, and IgG results for bacterial toxins of lipid polysaccharides were negative for any IgA, IgM, and IgG results for negative, Positive < / RTI > and IgG results for actomyosin are negative.

In relation to cross-cell pathway breakdown, diagnosis of intestinal wall collapse by bacterial antigens is based on the results of testing that any IgA, IgM, and IgG results for bacterial toxins of lipopolysaccharides are positive for all IgA , IgM, and IgG results are negative, and IgG results for actomyosin are negative.

Also, according to the discovery discussed herein, the diagnosis of disruption of both intestinal and blood brain barrier integrity by bacterial toxins is based on the test results that any IgA, IgM, and IgG results for bacterial toxins of lipopolysaccharides are positive, IgM, and IgG results for rhinidine and crude gel positive, for any IgA, IgM, and IgG results for blood brain barrier protein positive, and for any IgA, IgM, and IgG Diagnosed when the result is positive.

Conversely, a diagnosis of collapse of both intestinal and blood brain barrier by factors other than bacterial toxins was tested, with the result that the respective IgA, IgM, and IgG results for bacterial toxins of lipid polysaccharides were negative for acrolein, Any IgA, IgM, and IgG results positive, any IgA, IgM, and IgG results positive for blood brain barrier protein, and any IgA, IgM, and IgG results for neuronal cell antigens are positive do.

Also, intestinal bacterial imbalance in intestinal microflora can be caused by a gentle breakdown of the blood brain barrier rather than the intestinal barrier. For example, in this case, diagnosis of intestinal bacterial imbalance in intestinal microflora indicates that any IgA, IgM, and IgG results for bacterial toxins of lipopolysaccharide are IgA, IgM, and IgM for positive, And IgG results are negative, any IgA, IgM, and IgG results for the blood brain barrier protein are positive, and any IgA, IgM, and IgG results for neuronal cell antigens are positive.

Similarly, blood-brain barrier intact decay, neuroinflammation, and neuro autoimmune-related diagnoses, which are not associated with intestinal wall or intestinal bacterial intestinal bacterial imbalance, were tested for IgA, IgM, and IgE against bacterial toxins of lipopolysaccharides The IgG, IgM, and IgG results for the negative, ocuridine, and crude, IgG results are negative, any IgA, IgM, and IgG results for the blood brain barrier protein are positive, and any IgA, IgM, and IgG results are positive.

With respect to a particular disease, the test result analysis contemplated herein can be used to detect and assist in diagnosing amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, Alzheimer's disease, or peripheral neuropathy, major depression. This condition is diagnosed when test results show that any IgA, IgM, and IgG results for the blood brain barrier protein are positive, and any IgA, IgM, and IgG results for neuronal cell antigens are positive.

Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments together with the accompanying drawings and the tables.

Figure 1 shows the role of LPS in inducing leaky gut and brain leak syndrome.
FIG. 2 is a graph showing the role of LPS in inducing inflammation and Th17 lymphocyte activation in the pathogenesis of inflammatory and neuroimmune diseases, and induction of inflammatory response, cytokine production and Th17 positive cells of LPS.
FIG. 3 is a diagram showing an etiology of intestinal or brain function that can lead to mucosal tolerance loss when not treated, intestinal barrier dysfunction, systemic inflammation, nerve inflammation, nerve infiltration and neural degeneration.
Figure 4 is a diagram showing the proposed scenario of the present invention in which abnormal bowel permeability and blood brain permeability can be used for intestinal permeability identification (IPI) and / or blood brain permeability identification (BBPI).
Figure 5 is a diagram showing the role of the abnormal intestinal permeability proposed in the autoimmune disease pathogen according to certain aspects of the present invention.
Figure 6 is a view showing the placement of a sample microtiter plate for immunoassay, which is a 12 row microtiter plate with 12 different antigens and peptides according to a particular aspect of the invention.
Figure 7 is a view of the placement of a sample microtiter plate according to a particular aspect of the present invention, showing the presence of IgG / IgM < RTI ID = 0.0 > (IgG / IgM) < / RTI > for the twelve different antigens or peptides / IgA is measured.
Figure 8 is an illustration of the placement of a sample microtiter plate according to a particular aspect of the invention, wherein IgG / IgM / IgA is measured weekly (both bound antigen and peptide are clear) as negative and positive controls for qualitative control purposes.
Figure 9 compares IgG, IgM, and IgA for bacterial lipid polysaccharides and aclidine / crude lipid in healthy donors and gastroentero-autoimmune patients. Elevated IgG, IgM, IgA and IgG + IgM + IgG levels for bacterial lipid polysaccharides in healthy donors are indicated by bright bars in the left graph and dark bars in the case of gastric autoimmune patients with an average of more than 2 standard deviations . Elevated IgG, IgM, IgA and IgG + IgM + IgG levels for aclidine / cruderin in healthy donors are indicated by bright bars in the right graph and dark bars in the range of 2 standard deviations above the mean for gastric autoimmune patients do.
Table 1 shows, in accordance with a particular aspect of the present invention, IgG for IgG, IgM (IgG) against 12 different antigens representing environmental factor (LPS), intestinal and BBB proteins and related antigens in the first set of 3 healthy individuals , IgA antibody levels.
Table 2 shows, in accordance with a particular aspect of the present invention, IgG for IgG, IgM (IgG, IgG, IgG) against 12 different antigens representing environmental factor (LPS), intestinal and BBB proteins and related antigens in the second set of three healthy individuals , IgA antibody levels.
Table 3 shows, in accordance with a particular aspect of the present invention, IgG for IgG, IgM (IgG, IgM) against 12 different antigens representing environmental factor (LPS), intestinal and BBB proteins and related antigens in the third group of 3 healthy individuals , IgA antibody levels.
Table 4 shows, for certain twelve different antigens representing environmental factors (LPS), intestinal and BBB proteins and related antigens of three patients with chronic digestion disorder and intestinal permeability (Samples 10-12) IgG, IgM, and IgA antibody levels.
Table 5 shows, in accordance with a particular aspect of the present invention, IgG, IgM, IgA (IgG) for 12 different antigens representing environmental factor (LPS), intestinal and BBB proteins and related antigens of 3 patients Antibody levels.
Table 6 shows the results of immunoprecipitation of IgG, IgM (IgG) against 12 different antigens representing environmental factor (LPS), intestinal and BBB proteins and related antigens of MS patients 3 (Samples 10-12) , IgA antibody levels.
Table 7 shows a clinical analysis of specific antibodies to LPS, aclidine / choclrin and actomyosin network in the blood, according to certain aspects of the present invention.
Table 8 shows, in accordance with certain aspects of the present invention, a clinical analysis of elevated levels of specific antibodies to LPS, acridine / choclrin and actomyosin in saliva.
Table 9 shows the clinical interpretation of the elevation of specific antibody levels to LPS, occludin / chorine, blood brain barrier protein and neuronal cell antigens in blood, according to certain aspects of the present invention.

Increased antigen penetration is a prerequisite for disease outbreaks. It is known that in many conditions the intestinal permeability is increased and thus the antigen uptake is increased. IgA, IgM, IgG, and immunocomplexes are produced in the intestine in the form of IgA and IgM, and in the oocyte, respectively (26, 27).

I. Effect of bacterial toxins and inflammatory cytokines on blood-brain barrier agitation and induction of neuroinflammation

The blood-brain barrier (BBB) maintains internal environment and central nervous system stability. Structural and functional changes in BBB may be a cause of autoimmune diseases, especially autoimmune diseases such as multiple sclerosis (28).

The BBB is a parenchyma that typically causes necrotic cell death that can occur in response to environmental factors such as infection, toxins, excitotoxicity, or trauma, and blood leukocytes that respond normally to necrotic damage (23). The BBB consists of two layers. The first layer consists of vascular endothelial cells with abundant fusion membranes structurally similar to intestinal epithelial cells (24, 28). The second layer is the boundary of neurons (29), which are formed by glial proliferation of neurons. The perivascular space between endothelial cells and astrocytes is filled with macrophages that behave like immature dendritic cells (29). Thus, an element capable of opening the epithelial TJ barrier can destroy both the BBB and nerve tissue (30-33). This includes bacterial toxins, inflammatory cytokines, enzymes and effector cells Th1, Th17, and is essential for central nervous system inflammation (29, 34-35).

BBB decay by toxins, cytokines, chemokines, adhesion molecules, and others, and T-cell transport to the central nervous system in systemic compartments play an important role in the progression of MS lesions (36-38). However, when comparing human TH1 to Th17 lymphocytes, human Th17 lymphocytes pass BBB more rapidly than TH1 lymphocytes. Indeed, a number of IL-17- and IL-22-expressing CD4 + CD45RO + memory lymphocytes migrate through the BBB to express IL-17 + and IL-22 + markers, which can cross the BBB in vitro and in vivo, (35). BBB endothelial cells express IL-17R and IL-22R, which are used by Th17 lymphocytes to infect BBB endothelial cells (ECS). When IL-17 and IL-22 are added to a monolayer of human BBB-ECS, the spread of cells or antigens such as bovine serum albumin (BSA), macromolecules, etc. is greatly increased over BBB. This increased permeability of BBB-ECS correlates with decreased expression of two important fusion membrane proteins, aclidine and crude (39).

These results strongly suggest that inflammation caused by LPS and other bacterial toxins, which induce activation of IL-17 and IL-22 receptor expression in Th17 lymphocytes, may lead to Th17 and BBB fusion membrane binding in vascular-brain barrier endothelial cells. This disturbs the fusion membrane, resulting in transit of the BBB of Th17 and autoreactive T cells, interferon-gamma release by granzyme-B and CD4 cells by Th17 leading to neuronal cell destruction, release of neuronal antigen and BBB protein by circulatory system Cells that co-express IL-17, IL-22 and granzyme B in response to IL-17 and IL-22), BBB induction and destruction, BBB penetration into circulating CD4 + lymphocytes and soluble molecules (40-44). The role of Th17 lymphocytes in the pathogenesis of inflammatory and neuroimmune diseases is shown in Fig. Following this mechanism of action, intestinal permeable bacterial toxin induction and BBB protein structural disturbances lead to antibody production to fusion membrane proteins and BBB proteins, as well as to LPS. Thus, in accordance with certain aspects of the present invention, the challenge to neuroinflammation is disclosed as a test for LPS, acrodine, claudin, BBB protein, fusion membrane proteins, enzymes such as substrate metalloproteinase and related receptor antibodies, May lead to restoration of gastrointestinal barrier function abnormalities, systemic inflammation relief, and vascular-brain barrier recovery.

The expression of IL-17 and IL-22 receptors in blood vessel-brain barrier endothelial cells binds Thl7 cells to the BBB fusion membrane. The fusion membrane is thereby disturbed, leading to autoreactive CD4 cells and neural degeneration. Subsequently, Thl7 cells are traversed across the BBB, reaching the stage of neuronal necrosis with granzyme B release. This neuronal antigen release results in a malignant cycle of neuronal autoimmunity and neurodegeneration.

Based on the information presented here, it is assumed that the intestine is the starting point for many neurodegenerative diseases. Starting with an unbalanced microbial gun, it leads to the release of large amounts of lipopolysaccharide (LPS). Large amounts of LPS endotoxin upregulate proinflammatory cytokines TNF-alpha and IL-l beta leading to their proteolysis or detachment, including TJ and occludin and crude. The blood flow to the BBB then becomes inflamed. Inflammation opens the BBB, causing neuro-infiltration, nerve inflammation, nerve autoimmunity, and finally, neural degeneration. Figure 3 shows the pathophysiology leading to neurodegeneration; If the intestinal barrier function abnormality of the patient is not treated, it may develop into neurodegeneration with nerve inflammation and time course. Many autoimmune diseases involve multiple triggering, symptoms, and dysfunctions. In the case of neuronal autoimmunity, the immune system and the nervous system are involved in many patients who produce high levels of antibodies to the LPS, TJ, and BBB proteins. The common cause for these two systems is the GI, and its importance is mentioned (43).

Thus, in certain aspects of the present invention, antibody detection and measurement of TJ proteins such as ecludins, bacterial endotoxins such as LPS, and BBB proteins can be used to assess GI and intestinal wall integrity as well as systemic inflammation, neuroinflammation, It is the best way to determine and / or diagnose the root cause of neurodegeneration. In addition, any rectal epithelial lesions should be treated immediately. Otherwise, this is to allow the dietary protein, symbiotic and pathogenic bacteria to penetrate into the circulatory system, leading to inflammatory reactions leading to complex autoimmune and neuroimmune diseases.

II. Measurement of permeability of small sugar to large antigenic molecules

Current methods for intestinal permeability assessment involve the use of lactulose and mannitol. For the past 40 years, this has been a useful clinical method. The absorption of lactulose meant intestinal wall laceration and thus was considered intestinal permeability. Contrary to popular belief, the absorption of these small molecules is actually a microleakage rather than a thermal injury. Lactoolose is a relatively small molecule, and trans-membrane transport of these substances does not reflect food or other protein transfer and immune response situations to them. Furthermore, the lactulose / mannitol test measures only small molecules that pass around the cell and not the small molecule transfer through the cell crossing pathway.

Therefore, macromolecular intestinal permeability identification (LMIPI) should use macromolecules such as bacterial endotoxins (equivalent to dietary protein sizes) that are antigenic and threaten the immune system. Moreover, with respect to BBB permeability, changes in BBB permeability have not been recognized for blood testing for BBB permeability measurements, although there has been some increase (45-50), which is a major initiator in MS and experimental autoimmune encephalomyelitis (EAE). However, morphological and functional changes in BBB have been demonstrated using crude crushed / occludin to measure barrier impairment in animal studies (7, 11, 28).

As shown in FIG. 4, the method is focused on macromolecules that are antigenic and produce a specific IgG, IgM and / or IgA antibody that is threatening to the immune system when released from the barrier, Serum and / or saliva samples.

Assessment of intestinal barrier permeability to large antigenic molecules such as bacterial endotoxins and dietary proteins is important for understanding the pathogenesis of gastrointestinal and autoimmune diseases. There is a scientific increase that many gastrointestinal and autoimmune diseases are accompanied by increased bacterial toxin transport of aerobic and basic bacteria through endotoxins and other barriers (7, 51-55). Such increased transport and associated inflammation leads to fusion protein degradation and an immune response to fusion membrane proteins such as aclidine / crude liposomes and bacterial endotoxins such as LPS. In fact, exposed to bacterial toxin or gliadin-exposed rat and human epithelial cells secrete significant amounts of crude. This turbulent release leads to the protein ZO-1 separation in the fusion membrane complex, resulting in intestinal permeability through the peri-cellular pathway (7, 51). Many chronic diseases are associated with elevated serum IgA and IgM levels for LPS and other pathogenic bacterial antigens (24, 25). These conditions lead to intestinal inflammation and mucosal barrier permeability, which results in enlargement of the space between barrier cells and separation of the fusion membrane protein leading to the activation of the mitochondrial network and protective barrier. This loss of protective barrier increases bacterial delivery and thus increases serum levels of toxins, fusion membrane proteins, and actomyosin.

According to a particular aspect of the invention, elevated serum IgA and IgM for LPS, fusion membrane proteins (ecludin / choclrin) and actomyosin refer to intestinal barrier permeability and macromolecular transport through the barrier. Bacterial endotoxins can cause autoimmunity through bacterial toxins acting as superagents to T lymphocytes or through mechanisms called molecular mimicry. Many bacteria have antigenic sites very similar to human tissue antigens, including nerve tissue. If intestinal barrier permeability is left unconfirmed, the inflammatory response of the antigen and the antibody produced against it, this time, triggers autoimmunity, including neuronal autoimmunity, after inflammation in several tissues. Thus, antigen-presenting intestinal barrier permeability is allowed to proceed and continued degeneration leads to systemic inflammation, followed by induction of antigenicity and permeability of cellular blood-brain barrier permeability, and at the same time an additional immune response progresses, eventually leading to nerve inflammation, It is regressive.

Thus, in accordance with a particular aspect of the present invention, a patient having chronic inflammatory and autoimmune conditions measures IgA, IgG and / or IgM against bacterial LPS, fusion membrane protein and actomyosin to detect intestinal permeability to a large antigenic molecule . Finally, in addition to measuring IgA, IgG and / or IgM antibodies against LPS and acordin / joruline, patients with neuroimmune diseases also require that these antibodies to BBB proteins, enzymes, related receptors, and neuronal antigens be measured do. This multi-step TJ degradation by bacterial toxin and antibody production to TJ protein, LPS and tissue damage and other bacterial antigen release which can lead to autoimmunity are shown in FIG.

According to a particular aspect of the present invention, the detection and measurement of IgA and IgM in saliva against TJ protein and LPS and the detection and measurement of IgG, IgM and IgA in blood are the best analyzes of intestinal barrier function evaluation, / Crude and other fusion membrane proteins, as well as BBB protein and neuronal antibody (IgG, IgM and IgA) detection and measurement are the best methods for intestinal / BBB permeability and nerve autoimmune assessment.

Bacterial antigens (LPS) induce fusion membrane breakdown and crude release, inducing fusion membrane opening and passing through the fusion membrane of aocludin and LPS and successively into the submucosal layer, where occludin and LPS are expressed in macrophages and Presented to dendritic cells. Macrophages present these antigens to T and B cells; Followed by humoral (IgA, IgM and IgG antibodies to acrodine and LPS) and a cell-mediated aberrant immune response. This interaction between humoral and cell-mediated immunity ultimately carries an autoimmune process aimed at the intestinal epithelium and other tissue antigens, leading to typical tissue damage of autoimmune disease.

The following exemplary analyzes, uses, and evaluations for some test patients are described. Although other materials and methods similar or equivalent to those described herein may be applied to the practice or testing of the present invention, the preferred methods and materials will be described in the description to further illustrate the invention.

ELISA analysis

A. Materials and Methods - Plate and sample preparation:

E. coli 055: 85; Pseudomonas aeruginosa, Pseudomonas putida, Salmonella enteritidis, Salmonella typhimurium, Klebsiella pneumonia, Morganella morganii, Klebsiella pneumoniae, Escherichia coli K-235, Pseudomonas aeruginosa, Pseudomonas putida, Lactose, actomyosin, aquatic basic protein and a-B cristalline from Sigma / Aldrich (St. Louis, Mo.) were purchased from Sigma-Aldrich, Hafnia alvei, Citrobacter koseri. Glial glial fibrous acid protein (GFAP) was purchased from Boehringer Mannheim (Indianapolis, Ind.). In addition, the materials used were calcium-binding domain designated as S100-B in the present study as crude peptide 1, 2, 3, long ZOT receptor, herbal basic protein peptide 87-106, cell boundary protein, substrate metalloproteinase- , Brain ZOT binding protein-1, -2, calprotectin (MRP-8), and brain ZOT receptors, as well as the brain ZOT receptor, BBB-1 MSELEKAMVA LIDVFHQYSG REGDKHKLKK, BBB-2 SELKELINNE LSHFLEEIKE QEVVDKVMET, BBB-3 LDNDGDGECD FQEFMAFVAM VTTACHEFFE HE.

All peptide HPLC grades with purity greater than 90% were synthesized in EZ Biolab (Carmel, IN). Unless specifically stated to the contrary herein, all ranges set forth herein should be interpreted to include the endpoints, and the open range should be construed to include commercial values. All list values shall be considered to include intermediate values, unless the context clearly indicates otherwise.

The antigens and peptides were dissolved in methanol at a concentration of 1.0 mg / ml, diluted 1: 100 in 0.1 M carbonate-bicarbonate buffer, pH 9.5, and 50 ul, and added to each well of a bottom flat polystyrene ELISA plate .

Plates were incubated overnight at 4 ° C and washed three times with 200 ul of Tris-buffered saline (TBS) containing 0.05% Tween 20 (pH 7.4). 200 mL of 2% calf serum albumin (BSA) in TBS was added to prevent non-specific binding of immunoglobulins and incubated overnight at 4 占 폚. Plates were washed and quantified (QC) and stored at 4 ° C until use.

Enzyme conjugate included: affinity purified antibody phosphatase-labeled goat anti-human IgG (Jackson ImmunoResearch, Cat # 109-055-008); (Jackson ImmunoResearch, Cat # 109-055-011) and affinity purified antibody phosphatase-labeled goat anti-human IgM (Jackson ImmunoResearch, Cat. # 109-055-043) .

Other additional reagents and materials included in the methods described herein include: phosphate buffered saline powder (Sigma, Cat # P3813-10 PAK), calf serum albumin (Biocell, Cat # 3203-00) sodium azide Cat # S-5881), Magnesium Chloride (Sigma, Cat # S5516-500ML), Tween 20 (Sigma, Cat # P1379-1000ML), Glycerol (Sigma, Cat # D-8885), 1.0 N hydrochloric acid solution (Sigma, Cat # H3162-1GA), 5 mg substrate tablets: p-NPP (para-nitrophenylphosphate) Cat # S-0942) and distilled water (D. H ₂ O).

The microwell plate was coated with 12 different enteric-brain-associated antigens or peptides as shown in Fig. A calibrator and a positive control and patient diluted sample are added to the wells, and autoantibodies that recognize other antigens bind in the first incubation. After washing the wells to remove all non-binding proteins, the purified alkaline phosphatase-labeled rabbit anti-human IgG / IgM / IgA non-binding conjugate was removed in a further wash step.

The conjugate is visualized with a para-nitrophenyl phosphate (PNPP) substrate giving a yellow reaction product, and the intensity is proportional to the concentration of autoantibodies in the sample. Sodium hydroxide was added to each well to stop the reaction. The intensity of the color was read at 405 nm.

In some respects, a bright red or red tiger tower (SST tube) was used for specimen collection, although other sample collection devices could be considered in this analysis.

Blood samples were collected using sterile venous puncture technique and serum was obtained by standard procedures. In some aspects, a serum of at least about 100 ul, which corresponds to at least about 1 ml of blood for analysis, is preferred.

B. Test Analysis Procedure

Describe the IgG, IgM or IgA antibody assay procedure for LPS, intestine and / or BBB protein. In some embodiments, all reagents are allowed to reach room temperature prior to the start of the test assay. The test assay procedure comprises preparing a well or plate coated with the desired number and type of antigens and / or the desired number of peptides. When the microtiter wells are ready, 100 ul of a 1: 100 diluted control proofreader is added to rows A and B of the microtiter plate as shown in Figure 7 using a multi-channel pipette. As shown in FIG. 7, about 100 ul of a 1: 100 diluted patient test sample, here serum, is shown in rows C and D for clinical sample 1, in rows E and F for clinical sample 2, It is added to rows G and H in duplicate.

As shown in Fig. 8, a negative and positive control group was performed on a separate plate periodically (for example, every week) similar to clinical specimens.

Plates were incubated at room temperature for 60 min. After incubation, the moon was emptied and washed four times with PBS using an ELISA washer. 100 ul of the optimal diluted alkaline phosphatase-labeled goat anti-human IgA was added to the IgA plate or 100 ul of the enzyme-labeled IgG was added to the IgG plate in the optimal dilution state and the anti-IgM was added to the IgM plate in the optimal dilution state .

Each plate was incubated at room temperature for 30-60 min. Approximately 10 minutes before the end of the binding-incubation, 5 mg of p-nitrophenyl phosphate tablets were mixed with 5 ml of substrate buffer and mixed until the tablets were completely dissolved to prepare a substrate solution. The washing was repeated 4 times with PBS using an ELISA washer. Approximately 100 ul of the substrate solution was then added to each well l. Plates were incubated for 30 min at room temperature avoiding exposure to direct sunlight. The reaction was stopped by adding about 50 ul of 3N NaOH. The color intensity of the wells was read using a microtiter plate reader at 405 nm for the blank wells and the calibrator, control, and unknown sample absorbance values were recorded.

C. Calculation Result

After reading the plate at 405 nm to obtain an optical density value (OD ₄₀₅ ), the negative control OD, the positive control OD, and the OD of each clinical sample were divided by the calibrator average OD of rows A and B, (IV).

The index value (IV) for each antibody was calculated by dividing the mean OD of each double sample by the mean OD of the proofreader control values for twelve different antigens (e. G., The average OD of wells C1 and D1 in wells A1 and & Dividing the average OD of wells C2 and D2 by the average OD of wells A2 and B2 and dividing the average OD of wells C3 and D3 by the average OD of wells A3 and B3)

The result is compared with the set reference range.

Crude / Occludin Antigen Index Calculation

Cal 1 (OD) 0.48, Cal 2 (OD) 0.50, Sample 3A (OD) 3.4, Sample 3B (OD) 3.2

Index 6.7

D. Interpret the results

i. IgG / IgM / IgA antibody pattern in patients with chronic dyspepsia, gluten immunoreactivity and hypersensitivity, and Crohn's disease:

IgG for nine healthy individuals (Table 1-3) and three patients with chronic digestive disorder and intestinal permeability (Table 4), three patients with gluten impairment (Table 5), and three patients with multiple sclerosis (Table 6) , IgM, and IgA antibody patterns and comparisons are presented in Tables 1-6, respectively.

Chronic dyspepsia and gluten immunoreactivity / hypersensitivity / autoimmunity data interpretation and laboratory differences are presented in Table 7-9.

ii. IgG, IgM and IgA antibody patterns for chronic digestive dysfunction and intestinal permeability, gluten impaired patients, and intestinal, BBB protein and related antigens in MS patients.

Based on the calculated indices, we found that there were 9 healthy individuals (Table 1-3), 3 chronic digestive disorders and intestinal permeability patients (Table 4), 3 gluten phantom patients (Table 5), and 3 MS patients The IgG, IgM and IgA antibody patterns of Table 6 are presented in Tables 1-6, respectively. In all healthy individuals, antibody indices for other antigens are lower or very low (Table 1-3), except for LPS and MBP, which may have an antibody index greater than 1.5 but not significantly higher than 2.0.

As is evident in the case of deamidated a-gliadin 33-mer peptide, tissue transglutaminase (tTg), and IgG and IgA to the gliadin-tTg complex in patients with chronic digestive dysfunction, Different.

For example, in Table 4, in Table 10, these antibodies were significantly elevated for LPS, crude lipid / occludin, long ZOT receptor, cell boundary protein, MMP-3, alpha -B cristalline, This means that in addition to the increased intestinal permeability, the patient has BBB permeability. Sample 11 of Table 4 shows a significant antibody uptake to the cell border protein and long ZOT receptor and a gentle rise to LPS but not to the BBB protein and neuronal antigens, BBB permeability, neuronal autoimmunity, and potentially other autoimmunity.

IgG, IgM and IgA antibody levels for twelve different antigens representing intestinal to intracerebral in three gluten phantom patients (samples 13-15) are presented in Table 5. Glutathione peroxidase is identified by the presence of the deamidated? -Glyadine 33-mer peptide, tTg-2, gliadin-tTg complex, tTg-6 and IgG and IgA antibodies to the cerebellum antigen. In these patients, the antibody pattern is significantly higher for ZOT-binding protein, brain ZOT receptor, alpha-B cristalline, calprotectin, GFAP, and cell bound protein, which confirms barrier impairment.

Antibody levels for the 12 tested antigens in the three MS patients (Samples 16-18) are summarized in Table 6. In addition to abnormal MRI, MS diagnosis is possible with antibodies to MBP, Myelin spindle-cell glial cell glycoprotein (MOG), α-B cristalline, protein lipid protein, lymphocyte activation and proinflammatory cytokine production. Antibodies are detected at fairly high levels for neuronal antigen, BBB protein and crude crN / aclundine. This actually means that the MS patient is experiencing an abnormal BBB function.

iii. IgG, IgM and IgA Antibody Measurements for Bacterial Lipopolysaccharide and Oakurdine / Choline in Gastric Autoimmune Patients.

Intestinal permeability is severe in gastric autoimmune diseases (4). Figure 9 compares the antibody elevation to bacterial endotoxin (lipopolysaccharide) and fusion membrane structure (acrodine / crude) with healthy control and gastro-autoimmune patients.

Excessive entry of antigenic macromolecules through the intestinal epithelium leads to the development of multiple inflammatory cytokines and systemic inflammation and a gradual increase (56). Ultimately, triple factors leading to autoimmune diseases appear to be needed (genetic vulnerability, environmental exposure, and intestinal permeability).

According to a particular aspect of the present invention, antibody elevation to LPS, occludin / crude lymphocyte and actomyosin network is a biomarker that identifies healthy intestinal wall degradation and is associated with LPS, occludin / Antibodies to proteins along with neuronal antigens (eg, MBP, α-B cristalline, GFAP, calprotectin, and brain ZOT proteins) signify healthy intestinal barriers as well as BBB warming disruption.

According to certain aspects of the present invention, the clinical interpretation of elevated antibody levels for saliva LPS, aclidine / choclrin and actomyosin is presented in Table 8.

According to a particular aspect of the present invention, a clinical interpretation of elevated antibody levels for LPS, occludin / chorinol, blood brain barrier proteins and neuronal cell antigens in blood is provided in Table 9.

Case Study Examples

There are two cases of patients with chronic digestive dysfunction and multiple sclerosis.

A. Case Report # 1: Patients with Chronic Digestive Dysfunction and Intestinal Barriers

A 38-year-old woman, weighing 106 pounds and 5'4 "in weight, suffering from gastrointestinal disorders such as constipation, diarrhea and systemic pain and fibromyalgia-like syndrome and weight loss (1-2 pounds reduction every month for the last 6 months) Laboratory results Hemoglobin 9.9 g / dl, MCV 77 fL, erythrocyte sedimentation rate 54 mm / 1 hour and low levels of folic acid and vitamin B-12, but high levels of liver enzymes and high C-reactive protein showed abnormal CBC The detailed biochemical and immunological profiles, including ANA, rheumatoid factor, T3, T4 and TSH levels, were within normal range, with repeated GI discomfort, fever and headache, and a GI assessment. And immunohistochemical evaluations revealed a total villus atrophy of the Marsh III classification, at which point IgG and IgA concentrations for gliadin and transglutaminase were determined. And trans-glutamic 3-5 times higher than the standard range for both IgG and IgA Mina agents.

From the perspective of villi atrophy, I was diagnosed with gliadin and transglutaminase for the diagnosis of chronic digestive disorders. The patient initiated a blood transfusion with no anti-inflammatory drug and a no-gluten diet. After 3 months, overall GI discomfort improved and weight increased by 4 pounds, but CRP was still high, and body aches and fever continued. In this regard, and in order to identify the root cause of inflammation and insignificance, antibodies against LPS, crude lipid / occludin, and cell boundary proteins were examined. The results presented in Table 10 for Sample 10 were 3-6 times higher for the patient (Sample 10) than for the LPS, crude lipid / aclodine, IgG, IgM and IgA antibodies to cell boundary proteins, In addition to the symptoms, the patient is suffering from bacterial dislocation, fusion membrane damage and intestinal leak syndrome to large antigen molecules.

Thus, in addition to the non-gluten diet, the patient has treated the intestinal leak syndrome with live-lectin diets as well as live bacterial glutamine, N-acetylcysteine, EPA / DHA, vitamin D, lactoferrin, xylitol, and boswell acid. After 30 days of non-lectin and no-gluten diet, along with viable therapy, the patient's clinical condition improved significantly: the fever fell below 37 ° C and increased by an additional 6 pounds. After 60 days the treatment of intestinal leakage was reduced to only probiotics, but the non-gluten diet continued. After one year, all laboratory tests were repeated and the repeated test results for gliadin, transglutaminase, CRO, LPS, and crude / ochrodin were in the normal range, indicating that patients with chronic digestive disorders and intestinal leak syndrome This means that a non-gluten diet is effective in addition to managing leakage.

Discussion: The majority of patients with chronic digestive dysfunction are listed in the literature as patients with intestinal leak syndrome as well as villous atrophy. For this reason, only about 50% of patients with chronic digestive dysfunction are normalized after six months of treatment with a non-gluten diet. The mechanism by which the intestinal leak syndrome is induced in chronic dyspepsia is due to the binding of specific gliadin peptides to the epithelial cells and damage to the fusion membrane proteins in some individuals, which may be due to the fact that the cochlein / And released into the blood. In this particular case, some of the patient's symptoms improve to a no-gluten diet, but the no-gluten diet does not improve the inflammatory response induced by LPS transport and increased intestinal permeability. However, after 30-90 days (57-61) of natural remedies for fusion protein recovery along with no gluten diet, clinical symptoms and laboratory results returned to normal. Therefore, patients with chronic digestive dysfunction should be screened for intestinal leaks to antigenic macromolecules, and treatment for restoring intestinal barriers as well as chronic digestive disorders should be performed in parallel. The subject of the present invention is to provide such functionality.

B. Case Report # 2: Patients with multiple sclerosis, intestinal and brain-vascular barrier permeability

A 38-year-old, 38-year-old man, weighing 5'8 "in height, with progressive neck, back, and muscle pain for three weeks with limb weakness, was treated with neurology. Tightness on the torso and legs For more than two years, the patient was very depressed and did not receive any help from her. The overall history is that of vitamin B-12, an unidentified mild case of small red blood cell anemia treated with iron supplements There was no specificity.

A series of immunological profiles and neurological examinations have been initiated to clarify whether the patient is mild stroke and neurological or immune disorders.

Laboratory results showed normal chemistry and CBC as a hemoglobin result of 10.8 g / dL. Immunologic profiles such as ANA, rheumatoid factor, immune complex, total immunoglobulin, cardiac lipid antibody and thyroid function test were within normal range.

In an additional test, cerebrospinal fluid and blood samples were collected and all the tests were M. tuberculosis, Borrelia, CMV, EBV, herpes type-6, HTLV-1, -2 and syphilis. CSF protein was 0.7 g / L, and glucose was 2.3 mMol / L

Neurological examination showed normal ocular motility and lower left-eye and right-eye corrected visual acuity of 6/48 and 6/36. The patient was on a slight gait and had a vertebral weakness on both legs. The terminal test showed bilateral sensory levels below D10.

Magnetic resonance imaging (MRI) of the brain showed weak white matter abnormalities with weak general atrophy observed in MS patients.

AGA, tTg antibody and lactulose / mannitol test were conducted to investigate the possibility of gluten hypersensitivity, chronic digestive disorder and intestinal leak syndrome. IgG and IgA against transglutaminase were negative for chronic digestive disorder, although IgG and IgA anti - gliadin antibodies were all over the reference range 3-6 fold. Also, the lactulose / mannitol test results were very abnormal. Thus, the following additional tests were performed: LPS, crude collagen / occludin, intestinal ZOT receptor, cell bound protein, MMP-3, brain ZOT binding protein, brain ZOT receptor, calprotectin, GFAP, IgG, IgM and IgA antibody tests for lean, BBB protein, and MBP. The results summarized in Sample 17 in Table 6 show a significant increase in antibody levels to MBP and GFAP and confirm MS diagnosis consistent with abnormal MRI results. In addition, a significant elevation of antibody to crude / acyclovir, calprotectin, and BBB protein implies increased intestinal and BBB permeability of GI (Table 6). Based on these test results, we received intravenous 1 g methylprednisolone for 5 days and had clinical improvement. At this point, the patient received beta-ceron and showed significant improvement after 15 days. Nutrient glutamine, N-acetylcysteine, EPA / DHA, vitamin D, lactoferrin, xylitol, and Bosswell's acid were also administered with 200 mg of minocycline IV glutathione for the recovery of damaged BBB and intestinal disorders. Three months after this therapy, the overall health of the patient improved significantly.

It will be apparent to those skilled in the art that many modifications other than those described herein are possible without departing from the inventive concepts herein. Accordingly, the invention is not to be restricted except in the scope of the claims. Furthermore, in interpreting the specification and the claims, all terms should be interpreted as broadly as possible to match the context. In particular, "comprising" and "comprising" are to be interpreted as referring to an element, component or step in a non-exclusionary manner, Or < / RTI > utilized or combined. If the claim statement refers to at least one selected from the group consisting of A, B, C ... N, this sentence requires only one element of the group, not A and N, or B and N Should be interpreted.

references

Claims (27)

In a method for testing a sample derived from a human,
Measuring a first signal due to binding to a first portion of the sample and a bacterial toxin; And
Measuring a second signal due to binding to a second antigenic component selected from at least one of (a) a long-related antigen and (b) a blood brain barrier-associated antigen, Of the test method. 2. The method of claim 1, wherein the bacterial toxin comprises a lipid polysaccharide. 3. The composition of claim 2, wherein the native enteric-associated antigen is selected from the group consisting of (1) a long structural protein; (2) fusion membrane proteins; (3) a binding receptor for a fusion membrane protein; And (4) a cell boundary protein. 4. The composition of claim 3, wherein the blood brain barrier-associated antigen is selected from the group consisting of (1) blood brain barrier protein; (2) gliophage fibrous acid protein (GFAP); (3) matrix metalloproteinase (MMP); (4) brain ZOT binding protein; (5) brain ZOT receptor; (6) Calprotectin; And (7) a herbal basic protein. 3. The method of claim 2, wherein the intestinal structural antigen comprises Actin / Actomyosin. 3. The method of claim 2, wherein the fusion membrane antigen is selected from the group consisting of acridine and crude. 3. The method of claim 2, wherein the binding receptor comprises a long ZOT receptor. 3. The method of claim 2, wherein the structural protein comprises a substrate metalloproteinase-3 (MMP-3). The blood brain barrier-associated antigen of claim 1, comprising (1) a blood brain barrier protein; (2) gliophage fibrous acid protein (GFAP); And (3) a substrate metalloproteinase (MMP). In a method for diagnosing a disease associated with intestinal leak syndrome,
Ordering a laboratory test using the method of claim 1; And
And analyzing the results of the test. A method for diagnosing blood brain barrier permeability related disease,
Ordering a laboratory test using the method of claim 1; And
And analyzing the results of the test. In a test plate, the test plate has as its binding peptide a natural antigen consisting of (1) a bacterial toxin, and (2) at least one of (a) a long-related antigen and (b) a natural blood brain barrier-associated antigen. 13. The use of claim 12 wherein the enteric-associated antigen is selected from the group consisting of (1) a long structural protein; (2) fusion membrane proteins; (3) a binding receptor for a fusion membrane protein; And (4) a cell boundary protein. 14. The composition of claim 13, wherein the blood brain barrier-associated antigen is selected from the group consisting of (1) blood brain barrier protein; (2) gliophage fibrous acid protein (GFAP); (3) matrix metalloproteinase (MMP); (4) brain ZOT binding protein; (5) brain ZOT receptor; (6) Calprotectin; And (7) a herbal basic protein. 13. The method of claim 12, wherein the blood brain barrier-associated antigen is (1) a blood brain barrier protein; (2) gliophage fibrous acid protein (GFAP); (3) matrix metalloproteinase (MMP); (4) brain ZOT binding protein; (5) brain ZOT receptor; (6) Calprotectin; And (7) a herbal basic protein. A method for supporting diagnosis of an excess permeability-related disease of an anatomical barrier,
Obtaining a test result from an antibody test panel that generates a signal due to binding of the patient sample to the bacterial toxin and (a) a natural antigen selected from at least one of (a) a long-related antigen and (b) a natural blood brain barrier- step; And analyzing the test results. ≪ RTI ID = 0.0 > 11. < / RTI > 17. The method of claim 16, wherein the sample is a blood sample. 17. The method of claim 16, wherein the sample is a saliva sample. 17. The method of claim 16, wherein the step of analyzing the test results comprises the steps of: testing results showing that any IgA, IgM, and IgG results for bacterial toxins of the lipopolysaccharide are negative, all IgA, IgM, and IgG results for positive, , And the intestinal bacterial imbalance of intestinal micro-organism when the IgG result for actomyosin is negative. 17. The method of claim 16, wherein analyzing the test results comprises comparing the results of the test to the results of any of the IgA, IgM, and IgG results for the bacterial toxin of lipopolysaccharide, positive IgA, IgM, and IgG results for positive, Wherein the antigen is diagnosed as being associated with intestinal wall breakdown by bacterial antigens, through the periocentric pathway when the IgG results for negative, positive, and actomyosin are negative. 17. The method of claim 16, wherein analyzing the test results further comprises comparing all of the IgA, IgM, and IgG results for the bacterial toxin of the lipid polysaccharide to any IgA, IgM, and IgG results for negative, Wherein the IgG results for the positive, and for the actomyosin are negative, through the pericellular pathway, associated with non-bacterial antigen-induced intestinal collapse. 17. The method of claim 16, wherein the step of analyzing the test results comprises the steps of: testing all of the IgA, IgM, and IgG results for bacterial toxins of the lipopolysaccharide against all of the IgA, IgM, and IgG results for positive, Wherein the IgG results for the negative, and for the actomyosine are negative, via cross-cellular pathways, associated with intestinal wall breakdown by bacterial antigens. 17. The method of claim 16, wherein the step of analyzing the test results comprises the steps of: testing results showing that any IgA, IgM, and IgG results for the bacterial toxin of the lipopolysaccharide result in any IgA, IgM, and IgG results for positive, Positive IgA, IgM, and IgG results for positive, blood brain barrier proteins positive, and any IgA, IgM, and IgG results for neuronal cell antigens Positive for enteric and blood brain barrier A method of diagnosing a disease associated with both collapse. 17. The method of claim 16, wherein analyzing the test results comprises comparing the IgA, IgM, and IgG results for bacterial toxins of the lipopolysaccharide to any IgA, IgM, and IgG results for negatives, Positive IgA, IgM, and IgG results for positive, blood brain barrier proteins positive, and any IgA, IgM, and IgG results for neuronal cell antigens Positive For intestinal and blood due to factors other than bacterial toxins The brain barrier being diagnosed as involving collapse of both. 17. The method of claim 16, wherein the step of analyzing the test results comprises the steps of: testing results showing that any IgA, IgM, and IgG results for the bacterial toxin of the lipopolysaccharide result in respective IgA, IgM, and IgG results for positive, IgM, and IgG results for positive, negative, and negative blood brain barrier proteins are positive, and any IgA, IgM, and IgG results for neuronal cell antigens are positive. A method of diagnosing intestinal bacterial imbalance in intestinal microorganisms without malignant decay. 17. The method of claim 16, wherein analyzing the test results comprises comparing the IgA, IgM, and IgG results for bacterial toxins of lipopolysaccharides to IgA, IgM, and IgG results for negatives, IgM, and IgG results for the negative, blood brain barrier protein positive, and any IgA, IgM, and IgG results for the neuronal cell antigen were positive. Intestinal barrier or intestinal microorganism Total intestinal bacterial imbalance Irrelevant, blood brain barrier, gated disorder, nerve inflammation and neurological autoimmunity. 17. The method of claim 16, wherein the step of analyzing the test results comprises the steps of: testing results showing that any IgA, IgM, and IgG results for the blood brain barrier protein are positive, and any IgA, IgM, and IgG results for neuronal cell antigens are positive Wherein the disease is diagnosed as one of amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, Alzheimer's disease, or peripheral neuropathy, major depression.

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